Fig. 4. Developing neurons influence colonic organoids. (A) Tracking the expression changes of PHOX2B and NDRG4 in NCSCs and LMX1A in colonic organoids according to the co-culture period of colonic organoids and NCSCs. (B) Immunostaining images of NCSC-derived neurons expressing TUJ1 as a key difference between co-culture time points of 4 and 6 days. (C) Significant increase in substance P in the co-culture medium on day 6 compared day 4, as measured by ELISA. (D) Expression of colonic epithelial cell marker proteins of colonic organoid after substance P administration were validated by immunoblotting. (E) Scattering distribution of cells expressing CHGA in the colonic organoid was confirmed using immunofluorescence staining. (F) Presence of enteroendocrine cells detectable by 5-HT within the colonic organoid, and columnar epithelial cell morphology (red dotted line). (G) The expression of NK1R and NK3R mRNA in colonic organoids was validated using qRT-PCR, with or without treatment with substance P. (H) Protein expression levels of CHGA in colonic organoids were measured using ELISA after inhibition of NK1R with specific antagonists. Data expressed as mean±SD (n=s, biological repeat). p-values calculated by two-way ANOVA (A and C) or one-way ANOVA (G and H). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant. Scale bars=25 μm (B and E) or 10 μm (F). CO: colonic organoid, SP: substance P.

International Journal of Stem Cells 2023;16:269-80 https://doi.org/10.15283/ijsc23026

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